Epipodophyllotoxins are potent inhibitors of topoisomerase II, but are known to induce multidrug resistance (MDR) in the treatment of human cancers. Several 4beta- arylamino derivatives of 4'-0-demethylepipodophyllotoxin have been shown to overcome MDR in vitro at doses 10 times lower than those for clinically-used epipodophyllotoxins while retaining potent anti-tumor, topoisomerase and tubulin polymerization activity. The objectives of this project were to develop an analytical method for the assay of these derivatives in biological fluids and to define their pharmacokinetics in order to provide objective information to aid in the selection of the most appropriate candidates for preclinical development. An isocratic HPLC method was developed for assay of the derivatives employing a phenyl column, methanol/formate buffer mobile phase, and electrochemical detection. Pharmacokinetic profiles following intravenous injection of 20 micromoles/kg in mice, revealed that total body clearances were significantly lower for the nitro and ethylene dioxy derivatives (9 and 11 mL/min/kg, respectively) than for the hydroxy and fluoro derivatives (19 and 32 mL/min/kg, respectively) . This is indicative of comparatively higher plasma concentrations over time for the nitro and ethylenedioxy derivatives as a result of lower distribution in tissues and/or decreased elimination (excretion and metabolism). Plasma concentrations 3 hours following injection were approximately 1 micromoles for the nitro and ethylenedioxy derivatives but only 0.2 and 0.4 micromoles, respectively, for the hydroxy and fluoro derivatives.